High-resolution NMR structure of an RNA model system : the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined

High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2′-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined

Spektroskopische Untersuchungen zur Bestimmung von RNA-Ligand-Wechselwirkungen und RNA-Dynamiken

This thesis describes the structural characterization of interactions between biological relevant ribonucleic acid biomacromolecules (RNAs) and selected ligands to optimize the methodologies for the design of pharmacological lead compounds. To achieve this aim, not only the structures of the RNA, the ligand and their complexes need to be known, but also information about the inherent dynamics, especially of the target RNA, are necessary. To determine the structure and dynamics of these molecules and their complexes, liquid state nuclear magnetic resonance spectroscopy (NMR) is a suitable and powerful method. The necessity for these investigations arises from the lack of knowledge in RNA-ligand interactions, e.g. for the development of new medicinal drugs targeting crucial RNA sequences. In the first chapters of this thesis (Chapters II to IV), an introduction into RNA research is given with a focus on RNA structural features (Chapter II), into the interacting molecules, the biology of the specific RNA targets and the further development of their ligands (Chapter III) and into the NMR theory and methodologies used within this thesis (Chapter IV). Chapter II begins with a description of RNA characteristics and functions, placing the focus on the increasing attention that these biomacromolecules have attracted in recent years due to their diverse biological functionalities. This is followed by a detailed description of general structural features of RNA molecules. The biological functions of the RNAs investigated in this thesis (Human immunodeficiency virus PSI- and TAR-RNA and Coxsackievirus B3 Stemloop D in the 5’-cloverleaf element), together with their known structural characteristics are introduced in Chapter III. Furthermore, a description of the investigated ligands is given, focusing on the methods how their affinity and specificity were determined. The introduction is completed in Chapter IV, where the relevant NMR theory and methodologies are explained. First, kinetics and thermodynamics of ligand binding are summarized from an NMR point of view. Subsequently, a detailed description of the resonance assignment procedures for RNAs and peptidic ligands is given. This procedure mainly concentrates on the assignment of the proton resonances, which are essential for the later structure calculation from NMR restraints. The procedure for NMR structure calculation of RNA and its complexes follows with a short introduction into the programs ARIA and HADDOCK. The final part of this chapter explains the relaxation theory and the methodology to extract dynamic information from autocorrelated relaxation rates via the model-free formalism. In the Chapters V to VII of this thesis, the original publications are included and grouped into three topics. Chapter V comprehends the publications on the investigations of HIV PSI-RNA and its hexapeptidic ligand. These three publications[1-3] focus on the characterization of the ligand and its binding properties, its structure and the optimization of its composition aiming to improve its usage for further spectroscopic investigations.Die vorliegende Doktorarbeit behandelt die strukturelle Aufklärung von Wechselwirkungen zwischen biologisch relevanten Ribonukleinsäuren (RNA) und ausgewählten Liganden, sowie die Bestimmung der inhärenten Dynamik der RNA, um zur Methodenentwicklung für den Entwurf neuer Pharmaka beizutragen. Zur Bestimmung sowohl der Strukturen, als auch der Dynamiken stellt die Flüssig-Kernspinresonanz-Spektroskopie (NMR) eine ideale biophysikalische Methode dar. Die ersten Hälfte dieser Doktorarbeit gibt zum einen eine Einleitung in die RNA-Forschung mit besonderem Fokus auf den allgemeinen strukturellen und dynamischen Eigenschaften von Ribonukleinsäuren, stellt zweitens die ausgewählten RNA-Zielstrukturen und deren mit verschiedenen Methoden bestimmten Liganden vor, und erklärt drittens die zugrundeliegende NMR-Theorie und die verwendeten Methoden zur Untersuchung der Bindungs¬charakteristika, zur Strukturbestimmung der RNA und der Liganden und zur Ableitung dynamischer Parameter aus experimentellen Daten. Die zweite Hälfte dieser Arbeit ist der kumulative Teil und enthält die Originalpublikationen, die in drei Themenbereiche eingeteilt sind. Zuerst sind die drei Publikationen gruppiert, in denen die Bestimmung und Charakterisierung peptidischer Liganden der HIV Psi-RNA und deren Wechselwirkungen miteinander behandelt werden. Durch einen Phage-Display Assay wurde zunächst eine Konsensus¬sequenz eines peptidischen Liganden identifiziert (HWWPWW). Zur Verbesserung der Bindungseigenschaften wurde das Hexapeptid mittels einer Sequenzvariierung auf einer Membranoberfläche (SPOT-Assay) weiter optimiert (HKWPWW). Die weiteren strukturellen Untersuchungen der RNA-Ligand-Wechselwirkungen wurden per Fluoreszenz- und NMR-Spektroskopie durchgeführt, wobei die NMR-Spektroskopie aufzeigen konnte, dass das Peptid HKWPWW in zwei Konformationen der zentralen Prolinpeptidbindung zu beinahe gleichen Anteilen vorliegt. Die nächsten zwei Publikationen beschreiben die Ligandselektion gegen die Zielstruktur HIV TAR und die Strukturaufklärung des Komplexes mittels NMR-Spektroskopie. Als Liganden wurden Tripeptide synthetisiert, in denen zwei Arginine eine synthetische Aminosäure mit aromatischen oder hetero¬aromatischen Gruppierungen in ihrer Seitenkette flankieren. Mittels Fluoreszenz-Resonanz¬energietransfersichtung (FRET-Assay) wurde eine Vorauswahl der Liganden vorgenommen und die Interaktionen der ausgewählten Liganden mit der RNA per NMR-Spektroskopie konkretisiert. Eine intensive strukturelle Untersuchung des Liganden mit einer Pyrimidinylgruppe in der Seitenkette der zentralen Aminosäure in Komplex mit der TAR RNA ergab eine 2:1 Bindungsstöchiometrie des Liganden. Die erste stärkere Bindungsstelle im Bulge der RNA war bereits weitgehend bekannt als Ziel von Arginin-tragenden Liganden. Die strukturellen Untersuchungen konnten jedoch auch die zweite Bindungsstelle des Tripeptids unterhalb des Bulges lokalisieren. Zuletzt sind die zwei Publikationen zur Untersuchung der RNA-Dynamik zusammengefasst. Aus autokorrelierten Relaxationsraten der Kerne C1’ und C8 (für Purine) bzw. C6 (für Pyrimidine) in Nukleotiden der RNA Tetraloopsequenzen UUCG und CACG wurden mittels des Model-Free Formalismus Parameter abgeleitet, die über Dynamiken auf der Zeitskala von Pico- bis Nanosekunden der C-H Vektoren berichten. Die Verwendung optimierter und neuer Werte der C-H Bindungslänge und der Anisotropie der 13C-chemischen Verschiebung (13C-CSA) ermöglichte eine genauere Ableitung der inhärenten Dynamiken dieser RNA Moleküle. Diese Informationen konnten in die strukturellen Untersuchungen der glykosidischen Bindung durch kreuzkorrelierte Relaxationsraten eingebaut werden. Des Weiteren konnten die dynamischen Parameter bei verschiedenen Temperaturen mit Parametern abgeglichen werden, die aus Molekular-Dynamischen (MD) Trajektorien abgeleitet wurden. Dies ermöglichte die Visualisierung der internen Bewegungen zweier strukturell ähnlicher Tetraloops aus der YNMG-Familie, die sich aber in ihrer Stabilität unterscheiden. Bei Temperaturen nahe dem Schmelzpunkt des weniger stabilen CACG-Tetraloops offenbarten sich die Änderungen in der Dynamik, die zum Aufschmelzen des Loops führen

NMR-Untersuchungen zu dynamischen Umfaltungsprozessen in RNA-Molekülen

The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectroscopy. To understand the reactions is of considerable interest, because most biological active RNA molecules function by changing their conformation. This can be either an intrinsic property of their respective sequence or may happen in response to a cellular signal such as small molecular ligand binding (like in the aptamer and riboswitch case), protein or metal binding. The first part of the thesis (chapters II & III) provides a general overview over the field of RNA structure and RNA folding. The two chapters aim at introducing the reader into the current status of research in the field. Chapters II is structured such that primary structure is first described then secondary and tertiary structure elements of RNA structure. A special emphasis is given to bistable RNA systems that are functionally important and represent models to understand fundamental questions of RNA conformational switching. RNA folding in vitro as well as in vivo situations is discussed in Chapter III. The following chapters IV and V also belong to the introduction part and review critically the NMR methods that were used to understand the nature and the dynamics of the conformational/structural transitions in RNA. A general overview of NMR methods quantifying dynamics of biomolecules is provided in chapter IV. A detailed discussion of solvent exchange rates and time-resolved NMR, as the two major techniques used, follows. In the final chapter V of the first part the NMR parameters used in structure calculation and structure calculation itself are conferred. The second part of the thesis, which is the cumulative part, encompasses the conducted original work. Chapter VI reviews the general NMR techniques applied and explains their applicability in the field of RNA structural and biochemical studies in several model cases. Chapter VII describes the achievement of a complete resonance assignment of an RNA model molecule (14mer cUUCGg tetral-loop RNA) and introduces a new technique to assign quaternary carbon resonances of the nucleobases. Furthermore, it reports on a conformational analysis of the sugar backbone in this RNA hairpin molecule in conjunction with a parameterization of 1J scalar couplings. Achievements: • Establishment of two new NMR pulse-sequences facilitating the assignment of quaternary carbons in RNA nucleobases • First complete (99.5%) NMR resonance assignment of an RNA molecule (14mer) including 1H, 13C, 15N, 31P resonances • Description of RNA backbone conformation by a complete set of NMR parameters • Description of the backbone conformational dependence in RNA of new NMR parameters (1J scalar couplings) Chapters VII & VIII summarize the real-NMR studies that were conducted to elucidate the conformational switching events of several RNA systems. Chapter VIII gives an overview on the experiments that were accomplished on three different bistable RNAs. These molecules where chosen to be good model systems for RNA refolding reactions and so consequently served as reporters of conformational switching events of RNA secondary structure elements. Achievements: • First kinetic studies of RNA refolding reactions with atomic resolution by NMR • Application of [new] RT-NMR techniques either regarding the photolytic initiation of the reaction or regarding the readout of the reaction • Discovery of different RNA refolding mechanisms for different RNA molecules Deciphering of a general rule for RNA refolding methodology to conformational switching processes of RNA tertiary structure elements. The models for these processes were a) the guanine-dependent riboswitch RNA and b) the minimal hammerhead ribozyme. Achievements: • NMR spectroscopic assignment of imino-resonances of the hypoxanthine bound guanine-dependent riboswitch RNA • Application of RT-NMR techniques to monitor the ligand induced conformational switch of the aptamer domain of the guanine-dependent riboswitch RNA at atomic resolution • Translation of kinetic information into structural information • Deciphering a folding mechanism for the guanine riboswitch aptamer domain • Application of RT-NMR techniques to monitor the reaction of the catalytically active mHHR RNA at atomic resolution In the appendices the new NMR pulse-sequences and the experimental parameters are described, which are not explicitly treated in the respective manuscripts.Die vorliegende Doktorarbeit beschäftigt sich mit den strukturellen Änderungen in RNA Molekülen während dynamischer konformationeller Änderungen, die gemeinhin als RNA-Faltung bezeichnet werden. Im Gegensatz zur Proteinfaltung sind RNA-Faltungsprozesse nicht exklusiv als die Faltung einer definierten Konformation aus einem Ensemble an ungefalteten, d.h. ausgehend von unstrukturierten Molekülen, zu verstehen. RNA-Faltung beinhaltet vielmehr die strukturelle Umwandlung verschiedener stabiler Konformationen (die als RNA-Umfaltung benannt wird) und den Aufbau von molekularen Komplexen aus mehreren Molekülen (siehe Kapitel III). Die experimentelle Technik, die hier zur Untersuchung dieser Prozesse genutzt wurde, ist die hochauflösende Flüssig-NMR-Spektroskopie. Das Verständnis der strukturellen und biophysikalischen Grundlagen solcher Umfaltungsreaktionen von RNA ist essentiell, da solche konformationellen Änderungen die biologische Funktion der Moleküle modulieren. Dabei ist zu bemerken, dass eine Umfaltungsreaktion eine intrinsische Eigenschaft einer gegebenen RNA-Sequenz sein kann oder die Antwort auf ein externes zelluläres Signal, wie die Bindung eines niedermolekularen Liganden (z.B. in Aptameren und in Riboswitch RNAs), eines Proteins oder eines Metall-Ions. Der erste Teil dieser Doktorarbeit (Kapitel I & II) hält einen Überblick über die Themengebiete RNA-Struktur und RNA-Faltung bereit. Beide Kapitel führen in den derzeitigen Stand der Forschung ein. Kapitel II führt dabei entlang der hierarchischen Ordnung von RNA Molekülen und diskutiert die Eigenschaften von Primär-, Sekundär- und Tertiär-Strukturelementen. Ein besonderes Augenmerk wird dabei auf bistabile RNA Systeme gelegt; ihre wichtige biologische Funktionalität wird dargestellt, ebenso wird das Potential ausgeleuchtet, diese funktionale Klasse von RNA Molekülen als Modellsysteme zu nutzen, um fundamentale Fragen zu konformationellen Übergängen in RNA zu beantworten. In Kapitel III folgt sodann die Diskussion über RNA-Faltung in in vitro Experimenten als auch im zellulären Kontext (in vivo). Die Kapitel IV und V besprechen die NMR-spektroskopischen Techniken, die genutzt werden, um die Art und die dynamischen Eigenschaften von konformationellen/strukturellen Umwandlungen in RNA zu untersuchen. Hierbei wird der Schwerpunkt auf die verwendeten Techniken des Wasseraustauschs an labilen Protonen und der zeitaufgelösten NMR-Spektroskopie gelegt. Der zweite Teil der Doktorarbeit fasst kumulativ die durchgeführten Studien zusammen. Kapitel VI bespricht hierbei die grundlegenden NMR Techniken, die zur Strukturaufklärung von RNA Molekülen angewendet werden und zeigt deren Anwendungsmöglichkeiten an unterschiedlichen Beispielen von strukturellen und biochemischen Studien. Das folgende Kapitel VII beschreibt die komplette Resonanzzuordnung eines RNA Modell-Moleküls (14mer cUUCGg tetra-loop RNA) und stellt eine neue Pulstechnik vor, die zur Zuordnung der Resonanzen von quatären Kohlenstoffen in Purinbasen benützt werden kann. Weiterhin schließt sich ein Report an, wie die Konformation des Zuckerrückgrates in RNA-Molekülen bestimmt wird und schlägt mittels einer an oben genanntem Modellsystem durchgeführte Parametrisierung 1J skalare Kopplungen als neue Strukturparameter vor. Kapitel VII & VIII fassen die hierzu durchgeführten RT-NMR Studien zusammen. Kapitel VIII gibt hierbei einen Überblick über die Untersuchungen an drei bistabilen RNA-Systemen. Diese Moleküle wurden ausgewählt, da sie als Modelle für RNA-Umfaltungsreakionen dienen. Das finale Kapitel IX behandelt die Anwendung der oben ausgeführten neuen Methodologie auf konformationelle Umwandlungen von RNA Tertiär-Strukturelementen: a) Guanin-abhängige Riboswitch RNA (GSW) und b) Minimales "hammerhead" Ribozym (mHHR)

Development of new parameters for structure determination and dynamic investigations on biomacromolecules by NMR

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Includes bibliographical references.Nuclear magnetic resonance (NMR) spectroscopy is unique in the content of structural as well as dynamic information that it can provide at atomic resolution. The aim of this PhD-thesis was to contribute to the understanding of biochemical processes by means of NMR-spectroscopic techniques, targeting specific problems as well as contributing to the general understanding and providing new, widely applicable methods. The main focus was on the structural as well as dynamic study of ribonucleic acids (RNA). A new structural NMR method was developed aimed at the determination of the glycosidic torsion angle [chi] in RNA, which defines the relative orientation of the nucleobases in respect to the ribose moiety (Duchardt et al., 2004). [Chi] was derived from the angle dependence of carbon-hydrogen dipole-dipole, nitrogen chemical shift anisotropy cross-correlated relaxation rates (gamma-rates). Method development comprised the design of a novel NMR experiment, the [gamma](HCN), as well as the introduction of [gamma] versus [chi] parameterization curves. The novel method provides an accuracy of around 10 degrees or better, comparable to the precision of conventional angle determination techniques. In contrast to conventional methods, the [gamma](HCN) is sensitive to molecular size and will therefore proof beneficial in the investigation of larger RNAs by NMR Apart from this methodological contribution to RNA structure determination, the dynamic properties of the abundant YNMG RNA tetraloop motif (with Y=C or U; N= any base; M=C or A) were studied in a residue specific manner by means of ¹³C NMR relaxation measurements. The dynamics of the extraordinarily stable cUUCGg motif were compared to the less stable uCACGg hairpin, which forms the stem-loop D (SLD) in the regulatory 5'-cloverleaf of coxsackievirus 3B.(cont.) Measurements were carried out at 25⁰C, at which both motifs are stable, as well as at close to the melting point of SLD (43⁰0). Ribose and base moiety specific amplitudes and time-scales of motion were extracted from R₁, R₁-[rho] and the heteronuclear nOe of C₆ and C₁. in pyrimidines and C₈ and C₁. in purines by application of the model-free formalism (Lipari and Szabo, 1982). The application of the model-free formalism to C₁, and C₆ which possess an additional adjacent carbon spin, was examined for the uniformly isotope labeled RNA hairpins investigated in this study. In addition, the relaxation data analysis was optimized for the ¹³C chemical shift anisotropy based on chemical shift tensors available to date (Stueber and Grant, 2002;Fiala et al., 2000). While at room temperature, the dynamics closely follow the structural features of both hairpins, residues in the loop closing as well as in the adjacent base-pair exhibit highly increased flexibility at temperatures close to the melting point of SLD. In contrast, loop dynamics remain unperturbed. In SLD, the residues close to the loop are conserved among the family of rhino- and enteroviruses, indicating a sequence based mechanism of decoupling loop structure and stability in order to adjust to the twofold requirement of a defined structure for protein recognition and low stability to ensure efficient melting within the genomic transcription process. In addition to investigations on RNA, structural studies were also conducted on the [zeta]-chain of the T-cell receptor (TCR) in order to contribute to the elucidation of the early events in T-cell activation. According to earlier studies (Aivazian and Stem, 2000), the extracellular encounter of the T-cell receptor with an antigen is transmitted into the T-cell via a lipid induced structural transition of the TCR [zeta]-chain cytoplasmic domain.by Elke Duchardt.Ph.D

Ribosomal RNA dynamics studied by NMR-spectroscopy

The ribosome is a large macromolecular machine that consists of both ribosomal proteins and ribosomal RNA (rRNA). It is a complex consisting of two subunits held together by non- covalent interactions, intersubunit bridges and some of these bridging interactions are mediated by the rRNA. In this PhD-project the dynamics of such regions of rRNA, participating in intersubunit bridges and tertiary interaction within the rRNA have been investigated with solution state NMR- spectroscopy. These studies have been performed in the context of several miniaturized RNA systems, containing sequences of E. coli 16S rRNA. In particular, regions along helix 44 (h44), the penultimate stem of E. coli 16S rRNA have been studied. The stem-loop part of h44 has been studied in detail, this part of the rRNA contains a naturally occurring UUCG-loop and adenines participating in a tertiary interaction with helix (h8) in the 16S rRNA. In order to characterize the dynamics of these RNA constructs with NMR-spectroscopy, purified RNA material in large amounts is a necessity. Because of this we have developed an RNA-sample production method (Paper I) as well an NMR-experiment method (Paper II) that we call SELOPE, a method that can reduce the need of using isotopically enriched RNA material for NMR-studies. The first chapter of this thesis introduces the underlying theory for RNA-sample preparation as well as alternative techniques compared to the ones used in Paper I. In a similar manner the underlying theory for the NMR-technique is introduced and with some emphasis on concepts crucial for understanding the SELOPE experiment, to contextualize Paper II. The usage of NMR-spectroscopy for the measurements of dynamics in RNA molecules is also introduced. The first chapter of the thesis also includes a description of the ribosome to help further understanding of Paper III. In addition, during chapter 2-5 of this thesis some work related to 1H-R1r characterization of chemical exchange and cross-relaxation among RNA imino protons is described and discussed. In Paper I, the development of an RNA-sample preparation method is described. The method is based on in vitro transcription of the wanted RNA sequence followed by a HPLC-purification procedure that uses two different HPLC techniques for the purification, both Reverse Phase Ion Pairing (RP-IP) and Ion Exchange (IE) HPLC. The complete method offers a robust and versatile alternative to other RNA sample preparation methods such as preparative gel electrophoresis techniques. In Paper II, we describe the development of an NMR pulse sequence that utilize a homonuclear magnetization transfer block in unlabeled RNA molecules. The pulse sequence can then for instance be used to transfer NMR signal of unwanted signals to other spectral regions and can for instance be used to remove the signal of pyrimidine H6s from the region of H6/H8/H2 in RNAs. In Paper III, the work of characterizing the stem-loop part of E. coli 16S rRNA h44 is described. This work both shows a UUCG-loop with dynamics on a millisecond time-scale as well as the dynamical behavior of a group of unpaired adenine bases, the study of dynamics of these adenines could aid the understanding of tertiary interactions within rRNA

Études structurales par résonance magnétique nucléaire du ribozyme VS de Neurospora

Le ribozyme VS de Neurospora catalyse des réactions de clivage et de ligation d’un lien phosphodiester spécifique essentielles à son cycle de réplication. Il est formé de six régions hélicales (I à VI), qui se divisent en deux domaines, soit le substrat (SLI) et le domaine catalytique (tiges II à VI). Ce dernier comprend deux jonctions à trois voies qui permettent de reconnaître le substrat en tige-boucle de façon spécifique. Ce mode de reconnaissance unique pourrait être exploité pour cibler des ARN repliés pour diverses applications. Bien que le ribozyme VS ait été caractérisé biochimiquement de façon exhaustive, aucune structure à haute résolution du ribozyme complet n’a encore été publiée, ce qui limite la compréhension des mécanismes inhérents à son fonctionnement. Précédemment, une approche de divide-and-conquer a été initiée afin d’étudier la structure des sous-domaines importants du ribozyme VS par spectroscopie de résonance magnétique nucléaire (RMN) mais doit être complétée. Dans le cadre de cette thèse, les structures de la boucle A730 et des jonctions III-IV-V et II-III-VI ont été déterminées par spectroscopie RMN hétéronucléaire. De plus, une approche de spectroscopie RMN a été développée pour la localisation des ions divalents, tandis que diverses approches de marquage isotopique ont été implémentées pour l’étude d’ARN de plus grandes tailles. Les structures RMN de la boucle A730 et des deux jonctions à trois voies révèlent que ces sous-domaines sont bien définis, qu’ils sont formés de plusieurs éléments structuraux récurrents (U-turn, S-turn, triplets de bases et empilement coaxial) et qu’ils contiennent plusieurs sites de liaison de métaux. En outre, un modèle du site actif du ribozyme VS a été construit sur la base des similarités identifiées entre les sites actifs des ribozymes VS et hairpin. Dans l’ensemble, ces études contribuent de façon significative à la compréhension de l’architecture globale du ribozyme VS. De plus, elles permettront de construire un modèle à haute résolution du ribozyme VS tout en favorisant de futures études d’ingénierie.The Neurospora VS ribozyme catalyzes the cleavage and the ligation of a specific phosphodiester bond, which is essential for its replication cycle. It is formed of six helical regions (I to VI) that are divided in two domains: the substrate (SLI) and the catalytic domain (stems II-VI). The latter contains two three-way junctions that allow recognition of the stem-loop substrate in a specific manner. This unique mode of substrate recognition could be exploited to target folded RNAs for diverse applications. Even though the VS ribozyme has been extensively characterized biochemically, no high-resolution structure of the complete ribozyme has been published yet and this limits our mechanistic understanding. A divide-and-conquer approach was previously initiated to study the structure of the important subdomains of the VS ribozyme by nuclear magnetic resonance (NMR), but this approach needs to be completed. In this thesis, the structures of the A730 loop, the III-IV-V junction and the II-III-VI junction were determined by heteronuclear NMR spectroscopy. Moreover, a unique NMR approach was developed for localizing divalent metal ions, whereas several isotope-labeling strategies were implemented to facilitate the study or large RNA molecules. The NMR structures of the A730 loop and the two three-way junctions reveal that these subdomains are well defined, that they are formed by several recurrent structural elements (U-turn and S-turn motifs, base triples and coaxial stacking) and that they contain several metal-binding sites. Interestingly, structural similarities were identified between the VS and hairpin ribozymes, which allowed the modeling of the VS ribozyme active site. In summary, these studies significantly contribute to a better understanding of the global architecture of the VS ribozyme. In addition, they will allow the construction of a high-resolution model of the complete VS ribozyme and facilitate future engineering studies

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Approaches for studying RNA aptamers with molecular dynamics simulation

The objective of this dissertation is to study RNA aptamers with molecular dynamics simulation. It addresses fundamental challenges associated with RNA aptamers that can be investigated via molecular dynamics simulation, including the unavailability of 3D structures for the apo state, the challenge of ensuring good sampling for a flexible molecule, and the uncertainties that accompany molecular properties. The results presented in this dissertation focus on the application of multiple independent simulations to address these issues. I present results from multiple independent molecular dynamics simulations that are started from selected de novo predicted structures, according to experimentally determined base stacking, as a workflow to characterize the flexible apo state of an aptamer. I systematically investigate the sampling of multiple independent simulations by studying the nonlinear dynamic behavior, including principal component analysis and multivariate recurrent quantification analysis. I further propose a simulation assessment approach based on the root mean square deviation (RMSD) matrix eigenvalue and estimate molecular properties of interest with rigorous statistical analysis. I first develop a workflow that combines computational modeling and fluorescence experiments to study the structure and dynamics of the aptamer apo state. The selected predicted structures pass rounds of clustering and satisfy the stacking condition of critical bases in apo state determined from experiments. Multiple independent simulations from these selected structures effectively achieve better sampling than using the available NMR complex structure with ligand removed. It is also noticed that when the backbone is well aligned, a different base at the same position might also be potential binding site. This provides insight to the ligand binding mechanism, specifically, whether the flexible terminal loop adjust its whole structure or a critical base flips to fit the ligand. With the evidence that multiple molecular dynamics simulations can be used to investigate the conformation of aptamer for situations where a 3D structure is not available, I next investigate how well multiple independent simulations from different initial conformations sample the conformational space. The sampling of simulations started from different predicted structures is compared both qualitatively and quantitatively. The projection of sampled structures on selected principal components axes shows overlap among different groups of simulations as well as regions visited only by a specific group. The sampling of different groups of simulations is then further compared via recurrence quantification analysis using the top 10 principal components. The minimum length required for each independent simulation is determined. The number of independent simulations for sufficient sampling of the system is recommended based on the standard error of the mean for the molecular property of interest. Once the number of independent simulations and the minimum length of each simulation are known, it is necessary to systematically perform rigorous statistical analysis on any property of interest. Examination of the simulation quality can be done by looking at the progress of the largest eigenvalue from the RMSD matrix. Simulations or sections of simulations can be grouped as repeated measurements or enrichment, which further determines the uncertainty calculation. I recommend such a procedure because the sampling achieved with molecular dynamics simulations performed with limited timescales might display dependence on the initial conditions. This would lead to an outcome where different simulations could exhibit different error. I urge that care be taken in analyzing simulation outcomes and emphasize that taking the average is not sufficient